Preparation of a steroid compound



United States Patent 2,903,398. PREPARATION OF A STEROID COMPOUND DonaldA. Kita, Jackson- Heights, and Gilbert M. Shull, Huntington: Station,N.Y., assignors to Chas. Pfizer & (30., Inc., New York, N.Y., acorporation of Delaware No Drawing. Application February 4, 1954 SerialNo. 408,613 6 Claims. (Cl. 195-51);

invention is concerned witha novel process-for the preparation of A-androstadiene-SJ7-dione. More particularly it is concerned with aprocess. for preparing this compound by subjectingof a variety ofsteroid compounds to the enzymatic activity of a particular f ngus- Thisfungus was. received from the Quartermaster Corps and identified by themas a species of Pycnodothis (QM70 2');. A living culture of this;organism has been deposited with the American Type Culture Collection inWashington, DC, and given the number ATCC 1172-1.

It has been discovered that the enzymatic activity of this organism can.produce two types of changes in steroid molecules. The first is, theproduction of A rings which contain double bonds, at the l and 4positions, and keto groups at the 3 position. This change is broughtabout when a steroid containing a double bond at the 4 position and aketo group at the 3 position is subjected to the enzymatic activity ofthe organism.

Compounds containing the type of A ring shown in the starting materialinclude many well known steroids such as progesterone, testosterone,corticosterone and cortisone. There are also some indications that thistype of product results when the starting androstane steroid containsinstead a double: bond at the 5 position and an hydroxyl group at the 3position.

The second type of change is the production of D rings with oxygenatedgroups at the 17 position. The presence irr the product of a l7'-ketogroup has definitely been established andthere are indications that a17- hydroxyl group may instead be introduced in some. of the productmolecules. This type of change is produced when the starting steroidcontains any of a variety of groups at the 17 position. The originalgroup may be alkyl, including straight and branched hydrocarbon chainscontaining up to about 10'. carbon atoms, acyl, for example acetyl,benzoyl or other such groups derived from simple acids containing up toabout 10; carbon atoms, alkylene, including straight and branchedunsaturated hydrocarbon chains containing up to about 10 carbon atoms,COCH OH, COCH Oacyl, COCH O- al'kyl, etc. The starting steroid may alsocontain a double bond at the 16 position, but this is not necessary.

This product, A -androstadiene-3,17-dione is very valuable. Illustrativeof this value is the fact that it may readily be converted in one step,the well-known Inhoffen aromatization reaction, to estrone, a veryuseful compound.

It is to be understood that it is possible to carry out the processes ofthis invention, i.,e. subjecting a steroid compound to the enzymaticactivity of the fungus ATCC 11721, not only by means of a culture of theliving organism, but also by means of asuspension of mycelia from such aculture, orby means of the isolated enzyme systems, eitherintra-cellular or extra-cellular. These enzymes may be isolated bystandard procedures used in enzymol'ogy. It is often more convenient,however, to utilizethe living organism for the carrying out of theseprocesses. In general, it is preferred to grow the organism underconditions of submerged, aerobic fermentation, adding the steroid whichis to be treated at either the beginning of the fermentation or duringits course. The conditions for carrying out the fermentation may varyconsiderably. Sterile conditions should be employed throughout, and thetemperature is kept between about. 20. and. 35 C. for best results. Thecomposition of the nutrient medium may vary considerably. Ingeneral: asourceof carbohydrate is required, for example, glucose, sucrose orstarch. In addition, a source. of inorganic nitrogen, such as nitrates,ammonia salts, etc. is required. This. may be supplemented or replacedby an organic nitrogen. source, such as protein, hydrolyzate, soybeanmeal, cottonseed meal, etc. Inorganicsalts, particularly phosphates,potassium. salts, iron salts and magnesium salts, are desirable.

Throughout the fermentation, which on a large scale is run in vesselsequipped for submerged, aerobic fermentation, the medium is. agitatedand aerated at a rate of at least about /2 volume of air per volume ofmedium per minute. Occasionally it is necessary touse an anti-foamingagent, many of which are described in the technical literature, in orderto prevent excess foaming of the. mixture. In conducting the process ofthis invention, the steroid compound which is to be treated may be addedto the fermentation in finely divided solid form, or as a solution in asuitable nontoxic solvent, such as ethanol, acetone or isopropanol'.Preferably between about 0.01% and about 2% of the steroid by weight ofthe whole medium may be used. Although greater or lesser amounts may beused, there is no specific advantage. Addition of the compound may bemade at the time the fermentation medium is inoculated with the,microorganism, or the; compound may be added after growth of theorganism has, been established. Alternatively, the steroid compound maybe added. in small portions during the course of the, fermentation; Thecompound must be sterile in order that the mixture be not contaminatedby foreign microorganisms.

A further procedure that; has some value is the utiliza- 0 tion of; the.enzyme. systemsv formed during the growth of A specimen of the fungusATCC 11721 was inoculated into 100 ml. of a nutrient medium contained ina 300 ml. Erlenmeyer flask. This medium had the following composition:

, Percent Malt extract 5.0 Sucrose 1.0 Dipotassium ortho phosphate 0.1Sodium nitrate 0.2 Magnesium sulfate heptahydrate 0.05 Potassiumchloride 0.05

Ferrous sulfate heptahydrate 0.001 Distilled water, adjusted to pH 7.0

Fifty mg. of progesterone was introduced into the medium. The organismwas grown under aseptic, aerobic conditions at 28 C. The flask wasconstantly agitated by a rotary shaker.

After seven days, the mixture was extracted with 100 ml. of chloroform.The chloroform solution was evaporated to a volume of 2 ml., and asample of this solution was then subjected to chromatography onalumina-impregnated paper. The developing solvent was a mixture ofhexane and ether in the ratio of 15 to 5 by volume. In this way theproducts were first detected. The technique of paper chromatographywhich was used has been described by G. M. Shull et al. in Archives ofBiochemistry and Biophysics, vol. 37, p. 186 (1952). The system hereemployed is referred to in that paper as System III.

The final isolation of the products was accomplished by subjecting theabove-mentioned concentrated chloro form solution to chromatography on asilica gel-95% ethanol column. The developing solvent was a mixture of99 volumes of methylene chloride and one volume of 95% ethanol.

Five compounds were isolated. Some unreacted progesterone was firstrecovered. The next compound isolated was A -androstene-3,17-dione. ThenA -androstadiene-3,17-dione was recovered. This compound was present inby far the largest amount. Finally two unidentified steroids wereisolated. Although it has not been conclusively established, it isbelieved that one of these compounds is A-androstadiene-17-hydroxy-3-one. The physical constants of the A-androstadiene-3,l7- dione were as follows: melting point, 139140 C.

sulfuric acid chromogen maxima at 266, 305 and 370 m minima at 288 and350 m All these values are in excellent agreement with those previouslyreported in the literature.

Example II The experiment described in Example I was repeated, with nochange except the use of 16-dehydroprogesterone in place ofprogesterone. The products were isolated as in Example I, and once againthe main product was A androstadiene-3,l7-dione.

Example III When the same procedure is repeated, using 11-desoxy'corticosterone in place of progesterone, the main product 1s again A-androstadiene-3,l7-dione.

4 Example IV A -androstadiene-3,17-dione was again produced when thisprocedure was repeated, using A -androstene-3,17 dione as the startingmaterial. In this example, only the A ring was aflected, the D ring ofthe starting material being the same as that of the product.

The above examples are given for illustration only and are merelyspecific embodiments of this invention. Other obvious embodimentsinclude contacting the steroid with a suspension of mycelium obtainedfrom a culture of the organ-ism, or contacting the steroid with theenzymes isolated from the organism. The nutrient medium described aboveis conveniently used, but it is not a critical feature. Indeed, sincemany specific embodiments of this invention may be used withoutdeparting from the spirit and scope thereof, this invention is to belimited by the appended claims only.

What is claimed is:

l. A process for the preparation of A -androstadiene- 3,17-dione whichcomprises contacting with the enzymatic activity of the Pycnodothisfungus ATCC 11721 a steroid compound selected from the group consistingof:

R CH where R is a radical selected from the group consisting of where Ris a radical selected from the group consisting of alkyl, alkylene,acyl, --COCH OH, COCH Oacyl, and -COCH Oa1kyl.

2. A process as claimed in claim 1, wherein the starting steroid isprogesterone.

3. A process as claimed in claim 1, wherein the starting steroid is16-dehydroprogesterone.

4. A process as claimed in claim 1, wherein the starting steroid isll-desoxycorticosterone.

5. A process as claimed in claim 1, wherein the starting steroid is A-androstene-3,17-dione.

6. A process for the preparation of A -androstadiene- 3,17-dione whichcomprises cultivating the Pycnodothis fungus ATCC 11721 under submergedaerobic conditions at between about 20 and 35 C. in the presence of asteroid compound selected from the group consisting of:

R CH3 where R is a radical selected from the group consisting of alkyl,alkylene, acyl, --COCH 0H, -COCH Oacy1, References Cited in the file ofthis patent -COCHzOa1kY1, km, and hydroxyl; Ann. NY. Acad. Sci. 60, 1,p. 27.

( CH3 Bessey: Morphology and Taxonomy of Fungi, The

R Blakiston Company, Philadelphia, 1950, pp. 15-18. 0113A I 5 Bisby: AnIntroduction to the Taxonomy and Nomenclature of Fungi, 2d ed., 1953,The Commonwealth Myeological Institute, Kew, Surrey, pp. 3, 4, 29 to 33,75 and 117.

, Ainsworth & Cowan: Rules of Nomenclature for 10 Fungi and Bacteria,Jour. General Microbiology, 10, 0: 1954, pp. 465-474. Clements et al.:Genera of Fungi, 1931 (2d Printing where R is a radical selected fromthe group consisting 1954 Hafn p Co 1954, 13 to 15 and 353 of yl,alkylefle, W z 2 YL Ainsworth: A Dictionary of the Fungi, The Commonand-COCH Oa1kyl. 15 wealth Mycological Institute, Kew, Surrey, 1954, p.301.

1. A PROCESS FOR THE PREPARATION OF $1,4-ANDROSTADIENE3,17-DIONE WHICHCOMPRISES CONTACTING WITH THE ENZYMATIC ACTIVITY OF THE PYCNODOTHISFUNGUS ATCC 11721 A STEROID COMPOUND SELECTED FROM THE GROUP CONSISTINGOF: